The present invention relates generally to the field of compounds with antiangiogenesis effects that may be useful in the treatment of one or more neoplastic diseases.
In particular, the present invention relates to new and efficient methods of synthesizing prodrugs of the known antiangiogenesis compound denominated combretastatin A-4 and its analogs as described in U.S. Pat. Nos. 4,940,726; 5,409,953; and 5,569,786. More particularly, this invention relates to the improved and efficient phosphorylation and deprotection of phenol combretastatin A-4 in the synthesis of water soluble antiangiogenesis prodrugs of combretastatin A-4.
Combretastatin A-4 (Formula 1 below) is reported to be an antineoplastic compound inhibiting cancer cell growth and tubulin assembly. 
It is believed that combretastatin A-4 attacks the lining of blood vessels that grow around tumors, thereby severing the blood supply to the cancerous tumor. Although combretastatin A-4 has exhibited strong anti-cancer activity, its development has been inhibited by extremely poor solubility in water making development and biological distribution impracticable.
Water-soluble prodrug derivatives of combretastatin A-4 have been reported recently. In particular, synthesis of phosphate salts of combretastatin A-4, designated xe2x80x9ccombretastatin A-4Pxe2x80x9d (Formula 2 below) have been found to impart the requisite water solubility to the prodrug and are disclosed in U.S. Pat. No. 5,561,122 issued to G. R. Pettit et al. on Oct. 1, 1996. The phosphate group of the prodrug combretastatin A-4P reportedly is hydrolyzed in vivo to liberate the active drug combretastatin A-4. However, the currently disclosed methods for synthesizing combretastatin A-4P are difficult, require the use of undesirable solvents or restricted solvents, and are not easily scalable. 
where X=H(Z) (monovalent) or X=Z (divalent),
Z=Na2+, Na+, Li+, Mg2+, Mn2+, Zn2+, Ca2+, Cs2+, imidazol morpholine, piperazine, piperidine, pyrazole, pyridine, adenosine, cinchonine, glucosamine, quinine, quinidine, tetracycline, verapamil.
An improved method of preparing prodrugs of combretastatin is,necessary in order to meet the demand for an efficient and scalable synthesis to produce combretastatin A-4P and isomers thereof for effective use in treating cancer tumors and similar diseases.
It is an object of the present invention to synthesize prodrugs of combretastatin A-4 that are both water soluble and stable. It is a further object of the invention to develop an efficient and scalable method for synthesizing cis- and trans-prodrugs of combretastatin A-4.
Although combretastatin A-4 is a potent anticancer agent, its poor water solubility has hindered development of the drug as an anticancer treatment. Current methods of synthesizing water soluble derivatives of combretastatin A-4 require the use of undesirable or restricted solvents, such as chloroform, pyridine, dichloromethane or dimethylformamide (xe2x80x9cDMFxe2x80x9d), require extractions, separations and dilution of the reaction solutions, and heating and cooling of reaction mixtures at temperatures that are not suitable for production of prodrugs of combretastatin A-4 in commercial quantities.
As detailed herein, the subject invention provides a novel and improved method of synthesizing combretastatin A-4P that minimizes or eliminates the use of undesirable solvents, and overcomes many other deficiencies of the prior art using a continuous process. A novel process is herein disclosed in which dibenzyl phosphite/carbon tetrabromide is used to phosphorylate phenol combretastatin A-4 forming a phosphate ester of combretastatin A-4 with benzyl protecting groups thereon. An improved method of cleaving the benzyl protecting groups from the phosphate ester of combretastatin A-4 is disclosed in which bromotrimethylsilane is reacted with combretastatin A-4 to form phosphoric acid of combretastatin. An alternate novel phosphorylation process was concurrently developed and is herein disclosed in which bis(2,2,2-trichloroethyl) phosphorodichloridate phosphorylates combretastatin A-4 to a phosphate ester with trichloroethyl protecting groups thereon. The trichloroethyl groups are then cleaved from combretastatin A-4 using Zn/Cu amalgam to form a phosphoric acid of combretastatin A-4. Further improvements to the current processes for synthesizing phosphate salts of combretastatin A-4 are described herein disclosing a continuous process that overcomes many obstacles and limitations to the use and large scale production of combretastatin A-4 prodrugs.
In another aspect, the invention embraces the provision of combretastatin A-4 prodrug products of the aforesaid novel and improved method.
In a further aspect, the invention contemplates the provision of methods of synthesizing combretastatin A-4 prodrugs including a complete procedure for synthesizing cis combretastatin A-4, to which the foregoing method steps and procedures may then be applied to obtain the prodrug. The procedure for synthesizing cis combretastatin A-4 in accordance with this aspect of the invention includes the steps of obtaining a phosphonium salt of 3,4,5-trimethoxybenzyl bromide by mixing a brominating reagent and 3,4,5-trimethoxybenzyl alcohol in toluene to obtain the bromide, and adding triphenylphosphine thereto; obtaining tritylated isovanillin by mixing an amine base, isovanillin, and trityl chloride in an ether solvent, and after quenching, adding heptane and ethyl acetate; mixing a suspension of the phosphonium salt in tetrahydrofuran, an alkyl lithium reagent, and a slurry of the tritylated isovanillin, to obtain a cis/trans stilbene; and reacting the cis/trans stilbene with an acid to obtain a product consisting essentially of cis combretastatin A-4.
As in other embodiments of the invention, a combretastatin A-4 prodrug may then be prepared from the latter product by reacting the cis combretastatin A-4 with an activated phosphorylating agent having hydroxyl-protecting groups thereon wherein the phosphorylating agent is either dibenzylphosphite/carbon tetrabromide or bis(2,2,2-trichloroethyl) phosphorochloridate, to form a phosphate ester of combretastatin with protecting groups thereon; deprotecting the hydroxyl-protecting groups with a deprotecting agent to yield a phosphoric acid of combretastatin A-4; and reacting the phosphoric acid with reactive agent to form a phosphate salt of combretastatin A-4.
In the combretastatin A-4 synthesis procedure described above, it is currently preferred that the brominating reagent is phosphorus tribromide, the triphenylphosphine is unsubstituted triphenylphosphine, the amine base is triethyl amine, the ether solvent is tetrahydrofuran, the trityl chloride is unsubstituted trityl chloride, the alkyl lithium reagent is n-butyl lithium, and the acid is hydrochloric acid.
Further features and advantages of the invention will be apparent from the detailed description hereinafter set forth, together with the accompanying drawings.